The Expedit-Isotopomeric CrossLinking Mass Spectrometry (Expedit-ICLMS) technology for mapping global and dynamic protein-protein interaction networks

用于绘制全局和动态蛋白质-蛋白质相互作用网络的快速同位素交联质谱 (Expedit-ICLMS) 技术

• 批准号: 10377355
• 负责人: JEFFREY A RANISH
• 金额: $ 50.64万
• 依托单位: INSTITUTE FOR SYSTEMS BIOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377355
• 关键词: Address; Affinity; Architecture; Biological Models; Biological Process; Biomedical Research; Biotechnology; Cell Nucleus; Cell physiology; Cells; Complex; Complex Mixtures; Consumption; Crosslinker; Data; Databases; Development; Diagnostic; Disease; Effectiveness; Genetic; Genome; Goals; Health; Human; Individual; Knowledge; Label; Maps; Mass Spectrum Analysis; Methods; Molecular; Organism; Peptides; Play; Proteins; Proteome; Protocols documentation; Reagent; Reproducibility; Research Personnel; Resources; Role; Sampling; Signal Transduction; System; Technology; Therapeutic Intervention; Time; Yeasts; base; biological systems; cost; crosslink; effectiveness evaluation; experimental study; falls; gene regulatory network; improved; insight; interest; network architecture; new technology; novel; protein crosslink; protein protein interaction; searchable database; targeted treatment; technology development; technology research and development; yeast protein

SUMMARY This project seeks to develop new technology to enable global mapping of protein-protein interactions (PPIs) in a condition-specific, timely and affordable manner, by individual researchers interested in specific biological systems. Successful development of this technology is expected to have a transformative effect on all fields of biomedical research, directly addressing the purpose of the Focused Technology Research and Development R01 FOA (PAR-19-253). Global PPI networks are not only the most important resources for understanding the molecular mechanisms underlying normal and aberrant biological processes, but also the bases for understanding genetic interaction networks, constructing gene regulatory networks, and quantitative modeling of biological processes. Current technologies for mapping global PPI networks are labor intensive, time consuming, costly, plagued by false positives/negatives, and do not provide a way for individual researchers to efficiently map global PPI networks for particular biological systems. The proposed technology development is targeted to solve the two most important challenges facing crosslinking-mass spectrometry (CLMS)-based global PPI mapping studies: 1) the complexity of peptide mixtures derived from crosslinking samples with a large number of proteins and a large dynamic range of abundances, and 2) efficient and confident identification of crosslinked peptides by whole proteome database searches. We seek to overcome these challenges by developing a novel crosslinked peptide enrichment strategy, called Expedit, and combining it with the powerful capabilities of ICL crosslinkers, a new class of MS-cleavable, isotopomeric, bi- functional crosslinkers for crosslinked peptide identification. Unique features of ICLs permit 1) efficient determination of individual peptide masses in each crosslink from MS2 spectra, and 2) identification of crosslinked peptides by whole proteome database searching using a single MS2 spectrum per crosslinked peptide. The combination of Expedit with ICLMS is expected to address the major limitations of current CLMS approaches to enable routine large scale PPI studies for the first time. In the Aims, we will first synthesize novel Expedit reagents and evaluate their effectiveness for crosslinked peptide enrichment using increasingly complex mixtures. Once optimized, we will integrate Expedit with ICLMS and evaluate the effectiveness of the technology for building global PPI networks in yeast. We will evaluate the method in terms of the quantity and reproducibility of identified crosslinks/PPIs, the abundances of the identified proteins, their localization, affinities and complex membership (if available). We will compare our PPI networks to previously described yeast PPI networks. The effectiveness of the technology for crosslinked peptide identification will be evaluated by comparing it to state-of-the-art CLMS-based approaches. If successful, this project would provide a general and robust method for studying global PPIs and their dynamics that can be applied to any organism for which a sequenced genome is available.

概括 该项目旨在开发新技术，以实现蛋白质 - 蛋白质相互作用（PPI）的全球映射 对特定生物学感兴趣的个体研究人员，以特定条件，及时且负担得起的方式 系统。预计该技术的成功开发将对所有领域产生变革性影响 生物医学研究，直接解决重点技术研究的目的 开发R01 FOA（PAR-19-253）。全球PPI网络不仅是最重要的资源 了解正常和异常的生物学过程的分子机制，但也 理解遗传相互作用网络，构建基因调节网络和定量的基础 生物过程的建模。当前映射全球PPI网络的技术是劳动密集型的， 耗时，昂贵，受到误报/负面影响的困扰，并不为个人提供一种方式 研究人员有效地绘制特定生物系统的全球PPI网络。提出的技术 开发旨在解决交联质谱法面临的两个最重要的挑战 （CLMS）基于全局PPI映射研究：1）交联的肽混合物的复杂性 具有大量蛋白质和大量动态范围的样品，2）有效和 通过整个蛋白质组数据库搜索对交联肽的自信识别。我们试图克服 通过制定一种新颖的交联肽富集策略，称为Expedit和 将其与ICL交联的强大功能相结合，ICL交叉链接器是一类新的MS可辨别，同位素，双链接 用于交联的肽鉴定的功能交联。 ICL的独特功能允许1）高效 从MS2光谱中测定每个交叉链接中单个肽质量的确定，以及2）鉴定 通过每个蛋白质组数据库搜索的交联肽使用单个MS2光谱 肽。 Expedit与ICLM的组合有望解决当前CLM的主要局限性 首次启用常规大规模PPI研究的方法。 在目的中，我们将首先合成新颖的Expedit试剂，并评估其有效性 使用日益复杂的混合物交联的肽富集。一旦优化，我们将整合Expedit 使用ICLMS并评估该技术在酵母中构建全球PPI网络的有效性。我们将 根据已识别的交联/PPI的数量和可重复性评估方法， 在已识别的蛋白质中，其本地化，亲和力和复杂的成员身份（如果有）。我们将比较 我们的PPI网络以前描述了酵母PPI网络。该技术的有效性 通过将其与最先进的基于CLMS进行比较，可以评估交联的肽识别 方法。如果成功，该项目将为研究全球PPI提供一种一般而强大的方法 它们的动力学可以应用于可用测序基因组的任何生物体。